Abstract A central question in the field of Evo-Devo is how genes controlling embryonic development change during evolution. In insects, although the basic segmented body plan is highly conserved, our studies and others have revealed unexpected variation in the genes that control this process in different insect lineages. Here we will utilize comparative approaches, based on established arthropod phylogeny and the in-depth understanding of segmentation in the model insect Drosophila melanogaster, to explore the diversity of mechanisms promoting segmentation in insects. We will examine how re-wiring of regulatory circuits has occurred during evolution through the gain and loss of embryonic regulatory genes and changes in the interactions among them. We will build upon the establishment of non-model insect systems in our lab to carry out functional studies in diverse insect taxa to test the hypotheses that: (1) The pair-rule segmentation gene network is highly dynamic in insects; (2) Regulatory interactions between genes specifying segment formation and the upstream genes controlling them have been re-wired during evolution; and (3) Non-canonical pair-rule-like genes are required for segmentation in basally branching insects. These studies will contribute to our understanding of fundamental mechanisms regulating embryonic development and how these mechanisms have changed during the radiation of insects. This approach is novel in its systematic comparison of the regulatory mechanisms underlying a single biological process ? segmentation ? across species that diverged hundreds of millions of years ago. This project will train one postdoctoral fellow, four graduate students and at least three undergraduate students in molecular biology, genetics and molecular evolution.